The treatment of genetic and acquired immunodeficiencies by gene transfer is one of the most advanced field of gene therapy. Using the ZAP-70 protein tyrosine kinase deficiency as a paradigm, we previously demonstrated the feasibility of oncoretroviral-mediated hematopotetic stem cell (HSC) gene correction for ZAP-70-deficiency in a knock out mouse model (Steinberg/Otsu et al., Blood, 2002). While this approach has been successfully used in SCID patients, we hypothesized that some of the associated drawbacks may be overcome by in situ gene correction of T lymphoid progenitors in the thymus. In vivo intrathymic transfer of a gene providing a selective advantage for transduced prothymocytes should result in the generation of functional T lymphocyte progeny allowing long-term immune reconstitution. Indeed, following intrathymic injection of a T cell-specific ZAP-70-expressing lentiviral vector into thymi of ZAP-70-/- mice, we observed long-term (>12 month) differentiation of mature TCRap thymocytes in a subset of mice, indicating that the vector had integrated into progenitor cells. Moreover, peripheral ZAP-70-expressing T cells were generated in these mice, demonstrated a partially diversified receptor repertoire and were responsive to allo-antigens (Adjali et al., J Clin Invest, 2005). We now propose to optimize the efficacy and safety of this technology. We will improve delivery of virions to the thymus;notably, by injecting higher amounts of virions, performing repeat injections, and stimulating thymocyte proliferation. Additionally, we will improve the ZAP-70-expressing lentiviral vector (i) to enhance gene expression and specificity from the CD4 promoter, and (ii) to include safety measures such as HS4 insulator sequences and the HSV1-TK suicide gene. The immune reconstitution and function following intrathymic injections with these new methods and vectors will be compared with that obtained following ex vivo lentiviral-mediated correction of HSC. Ultimately, we will perform experiments using improved methods and vectors to assess T cell differentiation of human CD34+ derived cells from ZAP-70-deficient infants in NOD/Rag-/- mice. Altogether, the data emerging from these experiments should promote the development of an optimal and safe gene therapy strategy for patients with ZAP-70-deficiency as well as other forms of SCID.